兔抗人唾液酸转运蛋白抗体的制备及特性鉴定

Preparation and identification of the rabbit antibody against human sialin

目的：制备兔抗人唾液酸转运蛋白（sialin）的抗体，并进行特性鉴定。方法：应用RT—PCR从培养的人颌下腺细胞系HSG中扩增编码sialin N端1～38氨基酸的DNA序列，构建重组表达质粒pGEX-5X-1-sialin，测序鉴定后，转化大肠杆菌JM109，在0．1 mmol／L IPTG的诱导下表达GST—sialin融合蛋白。经SDS-PAGE鉴定后，利用GSTrap FF^TM柱纯化融合蛋白，并以其免疫家兔制备抗人sialin抗体。用ELISA法测定兔抗sialin血清的效价；用Western blot及免疫细胞化学等方法鉴定抗血清的特异性。结果：构建了重组表达质粒pGEX-5X-1-sialin；以其转化大肠杆菌在IPTG诱导下，获得以可溶性形式表达的GST-sialin融合蛋白；表达的GST—sialin经GSTrap FF^TM柱纯化后，免疫家兔制备出抗sialin抗血清，ELISA法测定抗血清的效价为1：32000。Western blot鉴定表明，制备的抗sialin抗体可特异地识别HSG细胞中相对分子质量（Mr）约55000的sialin蛋白。免疫细胞化学检测表明，sialin抗血清识别的抗原定位于HSG细胞的细胞质和胞核。结论：成功地制备出兔抗人sialin抗血清，为进一步研究sialin在涎腺组织的功能奠定了基础。

AIM： To prepare the rabbit antibody against human sialin and identify its properties. METHODS： Recombinant expresssion vector pGEX-5X-1-sialin was constructed, in which the sialin cDNA encoding the 1 -38 aa was fused to the C-terminal of the gene encoding the GST protein. The GST-sialin （N1 -38 ） fusion protein was expressed in E. coli JM109 at 37℃ in the presence of IPTG at 0. 1 mmol/L for induction for 3 hours, purified by GSTrap FF^TM, and then used as the immunogen to prepare the rabbit polyclonal antibody. The properties of antiserum against human sialin were identified by ELISA, Western blot and immunocytochemistry. RESULTS： The recombinant expression plasmid pGEX-5X-1-sialin was constructed. The GST- sialin （N1 -38 ） fusion protein was highly expressed with a molecular weight of 30 kDa, and the yield of the fusion protein was about 20% to 30% in total E. coli protein. The Utre of antiserum against human sialin was 1 ： 32 000. Western blot analysis proved the rabbit polyclonal antibody could identify both GST-sialin （ N1 - 38 ） fusion protein and GST. Besides, it specially recognized a 55 kDa band expressed in the human submandibular gland （HSG） cell line. The antigen recognized by the antibody was located in the cytoplasm and nucleus of HSG cell. CONCLUSION： The successful preparation of the polyclonal antibody against human sialin will provide efficient affinity reagent for further functional study of sialin expressed in human salivary glands.